Cellular DNA undergoes double strand breakage during the course of many physiological events as well as in response to a variety of environmental insults (Friedburg, E., Walker, G. & Siede, W., DNA Repair and Mutagenesis, ASM Press, Washington DC, 1995; Nickollof, J. & Hoekstra, M., DNA Damage and Repair, Humana Press, Totowa, N.J., 1998). Left unrepaired, such double strand breaks (DSBs) lead to mutations that may prove lethal to the organism. Therefore, these DSBs are repaired promptly via two independent pathways: i) homologous recombination; or ii) non-homologous end joining (Friedburg, E., Walker, G. & Siede, W., DNA Repair and Mutagenesis, ASM Press, Washington D.C., 1995; Nickollof, J. & Hoekstra, M., DNA Damage and Repair, Humana Press, Totowa, N.J., 1998).
The first pathway involves a series of very specific biochemical reactions catalyzed by a complex of cellular proteins (Shinohara & Ogawa, Trends in Biochem. Sci. 237: 387–391, 1995). Due to the large number of proteins involved in this complex, it is referred to as a ‘recombinosome’ (Hays et al., Proc. Natl. Acad. Sci. USA 92: 6925–6929, 1995). This pathway is the dominant mode of DSB repair in lower eukaryotes such as yeast (Nickollof, J. & Hoekstra, M., DNA Damage and Repair, Humana Press, Totowa, N.J., 1998). Therefore, yeast has been used as a model eukaryote to study the biochemical and molecular details of the double strand break repair. RAD51 is one of the genes of the RAD52 epistasis group that is involved in this pathway. This gene encodes a protein (Rad51) of about 38 kDa. Rad51 is a structural and functional homologue of the bacterial recombinase enzyme (also known as RecA).
Because of the crucial role of double strand break repair pathways in maintaining genomic stability, they have been found to be conserved throughout evolution. Consequently, RAD51 homologues have been discovered and characterized from many animal and plant sources (reviewed by Ogawa et al. In Cold Spring Harbor Symp. On Quant. Biol., Vol. LVIII pp. 567–576, 1993). Moreover, many eukaryotes have multiple forms of the RAD51 gene. The RAD51 family also includes structurally and functionally related genes such as DMC1, LIM15, RAD55 and RAD57. DMC1 has been implicated in meiotic recombination and associated double strand breaks (reviewed by Ogawa et al. In Cold Spring Harbor Symp. On Quant Biol., Vol. LVIII pp. 567–576, 1993).
All the members of the RAD51 family and their bacterial counterpart (RecA) share an important structural motif known as the ‘RecA signature sequence’ or Domain II. This sequence forms the ATP binding sites, an important property of all these proteins. However, the eukaryotic members of the RAD51 family can be distinguished from the bacterial RecA protein by the presence of an N-terminal extension present only in the RAD51 family members and a C-terminal extension of about 100 amino acids that is present in RecA but not in RAD51 family members. Important differences also exist in the primary structure of eukaryotic RAD51, and other closely related genes such as RAD55 and RAD57, indicating different physiological roles for each of these genes (Johnson, R. D. & Symington, L. S., Mol. Cell. Biol. 15: 4843–4850, 1995). It has been suggested that protein products of RAD55 and RAD57 genes interact with the RAD51 gene product during double strand break repair and recombination. The RAD57 gene of the budding yeast Saccharomyces cerevisiae also belongs to the RAD52 epistasis group (Kans, J. & Mortimer, R. Gene 105: 139–140, 1991). All members of this group are required for double strand break repair and genetic recombination. The RAD57 gene encodes a protein (Rad57) which shows significant homology to bacterial RecA and the eukaryotic counterpart, the Rad51 protein (Jeggo, P. Radiat Res. 150: S80–S91, 1998). Specifically, the ATP-binding motif (Walker Box A) is conserved in all the known Rad57 sequences (Hays, S. et al. Proc. Natl. Acad. Sci. 92: 6925–6929, 1995). Functional analysis has revealed interactions of Rad57 with Rad51, Rad52 and Rad55 to form a ‘recombinosome’ (Johnson, R. et al., Mol. Cell. Biol. 15: 4843–4850, 1995). Furthermore, in yeast, the Rad57-Rad55 heterodimer markedly stimulates DNA strand exchange by Rad51 (Sung P. Genes Develop. 11: 111–1121, 1997).
Biochemical studies have established that Rad51 catalyzes the strand exchange reaction between a circular ssDNA and linear dsDNA in presence of ATP and Mg+2 ions (Sung, P. Science 265: 1241–1243, 1994; Sung, P. & Robberson, D. L. Cell 82: 453–461, 1995). These properties are very similar to RecA. However, unlike the bacterial protein, strand exchange by Rad51 is slower and requires the presence of 3′ and 5′ overhangs that are complementary. Rad51 does not promote joint molecule formation if the linear DNA has blunt or recessed complementary ends. As a consequence of this requirement for the presence of overhangs, the Rad51 catalyzed strand exchange reaction has a polarity (Sung, P. Science 265: 1241–1243, 1994; Sung, P. & Robberson, D. L. Cell 82: 453–461, 1995). Also, whereas RecA binds to ssDNA or partially single-stranded DNA, Rad51 shows similar binding affinity for ssDNA and dsDNA. These biochemical observations, coupled with extensive genetic studies indicate the involvement of additional proteins in the eukaryotic recombination and double strand repair reactions.
Recent studies have uncovered several RAD51 genes in higher eukaryotes (Vispe, S. & Defais, M., Biochimie 79: 587–592, 1997). For example, at least four human RAD51 genes, RAD51 (Yoshimura, Y., et al., Mol. Cell. Biol. 21, 1665.), RAD51B/REC2 (Rice, M. C., et al., Proc. Natl. Acad. Sci. 94: 7417–7422; Albala, J. S., et al., Genomics 46, 476–479, 1998), RAD51C (Dosanjh, M., et al., Nucleic Acid Res. 26: 1179–1184, 1998), and RAD51D (Pittman, D. et al., 49: 103–111, 1998) have been cloned and characterized.
Existence of multiple isoforms of RAD51 gene products in higher eukaryotes suggests their differential functional role in meiotic versus mitotic recombination. Interestingly, while the yeast RAD51 is not an essential gene, the mouse RAD51 null mutations are embryonic lethal (Tsuzuki, T., et al., Proc. Natl. Acad. Sci. 93: 6236–6240, 1996). Biochemical and genetic analyses of different orthologs of RAD51 (preferably from the same species) will shed light on their precise functions. Nonetheless, two very recent studies have clearly established that overexpression of Rad51 protein stimulates homologous recombination and increases resistance to ionizing radiation in immortalized human cells (Xia, S. et al., Mol. Cell. Biol. 17: 7151–7158, 1997) and Chinese hamster cells (Vispe, S., et al., Nucleic Acid Res. 26: 2859–2864, 1998).
In view of the central role of RAD51 gene products in recombination and double strand repair, RAD51 genes from maize find use as a tool for improving maize transformation in general and maize gene targeting in particular. The present invention describes full-length cDNAs for a novel maize ortholog of RAD51, which shows high homology to the human RAD51C gene.
Control of homologous recombination by modulating RAD51 provides the means to modulate the efficiency with which nucleic acids of interest are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.